PROJECT SUMMARY The overall goal of this project is to improve our understanding of the acute effects of alcohol on G-protein coupled receptor (GPCR) signaling. Acute alcohol use produces profound changes in the signaling of many GPCRs in the brain, which coincide with diverse neurological responses to alcohol, including memory impairment, reward and loss of coordination. Because these molecular responses to alcohol are a key predictor of vulnerability to alcohol use disorders, it is crucial to better understand how GPCR signaling is modulated in response to alcohol. One potential regulator of ethanol-induced alterations in GPCR signaling is membrane cholesterol because acute alcohol exposure alters membrane cholesterol homeostasis. In both rat prefrontal cortex (PFC) and neuroblastoma N2A cells stably expressing 5-HT1A receptors (5-HT1ARs), I found that ethanol increased both membrane cholesterol content and 5-HT1AR-mediated G-protein signaling. In parallel, I found that ethanol treatment increased partitioning of 5-HT1AR/G-protein into lipid rafts of N2A cells, suggesting that cholesterol regulates receptor/G-protein translocation into lipid rafts. In this NRSA proposal, I will test the central hypothesis that membrane cholesterol regulates ethanol-induced increase in 5-HT1AR- mediated G-protein signaling by facilitating compartmentalization of 5-HT1AR, G?i3 and adenylyl cyclase (AC) into lipid rafts. I will test this hypothesis through two Specific Aims. In Aim 1, I will use simvastatin, an inhibitor of HMG-CoA reductase involved in cholesterol synthesis, to deplete brain cholesterol and measure 5-HT1AR- mediated G-protein signaling in rat PFC after acute ethanol exposure. In Aim 2, I will employ a N2A cell model to determine the effect of cholesterol depletion on compartmentalization of 5-HT1AR, Gai3 and AC in lipid rafts after acute ethanol exposure. Collectively, these studies will highlight membrane cholesterol as an important factor contributing to alcohol abuse through its regulation of alcohol-induced alterations in GPCR compartmentalization and signaling and identify cholesterol as a potential target for more effective treatment of alcohol use disorders.